Calpain-DependentDegradationofNucleoporins ...e3218f4d-53af-4b9b-83f0-027ba6492fbc... · mer’s disease and ALS (Fontana, 2015; Miladinovic et al., 2015). ... and further fixed with

Neurobiology of Disease

Calpain-Dependent Degradation of NucleoporinsContributes to Motor Neuron Death in a Mouse Model ofChronic Excitotoxicity

X Kaori Sugiyama,1* Tomomi Aida,1* Masatoshi Nomura,2 Ryoichi Takayanagi,2 XHanns U. Zeilhofer,3,4

and X Kohichi Tanaka1,5,6

1Laboratory of Molecular Neuroscience, Medical Research Institute, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8510, Japan,2Department of Medicine and Bioregulatory Science, Graduate School of Medical Science, Kyushu University, Higashi-ku, Fukuoka 812-8582, Japan,3Institute of Pharmacology and Toxicology, University of Zurich, 8057 Zurich, Switzerland, 4Institute of Pharmaceutical Sciences, Swiss Federal Institute ofTechnology Zurich, 8092 Zurich, Switzerland, 5Center for Brain Integration Research (CBIR), TMDU, Bunkyo-ku, Tokyo 113-8510, Japan, and 6CoreResearch for Evolutional Science and Technology, Japan Science and Technology Agency, Sanbancho, Chiyoda-ku, Tokyo 102-0075, Japan

Glutamate-mediated excitotoxicity induces neuronal death by altering various intracellular signaling pathways and is implicated as acommon pathogenic pathway in many neurodegenerative diseases. In the case of motor neuron disease, there is significant evidence tosuggest that the overactivation of AMPA receptors due to deficiencies in the expression and function of glial glutamate transporters GLT1and GLAST plays an important role in the mechanisms of neuronal death. However, a causal role for glial glutamate transporter dysfunc-tion in motor neuron death remains unknown. Here, we developed a new animal model of excitotoxicity by conditionally deletingastroglial glutamate transporters GLT1 and GLAST in the spinal cords of mice (GLAST �/�/GLT1-cKO). GLAST �/�/GLT1-cKO mice(both sexes) exhibited nuclear irregularity and calpain-mediated degradation of nuclear pore complexes (NPCs), which are responsiblefor nucleocytoplasmic transport. These abnormalities were associated with progressive motor neuron loss, severe paralysis, and short-ened lifespan. The nuclear export inhibitor KPT-350 slowed but did not prevent motor neuron death, whereas long-term treatment of theAMPA receptor antagonist perampanel and the calpain inhibitor SNJ-1945 had more persistent beneficial effects. Thus, NPC degradationcontributes to AMPA receptor-mediated excitotoxic motor neuronal death, and preventing NPC degradation has robust protectiveeffects. Normalization of NPC function could be a novel therapeutic strategy for neurodegenerative disorders in which AMPA receptor-mediated excitotoxicity is a contributory factor.

Key words: animal model; excitotoxicity; glutamate; motor neuron; transporter

IntroductionGlutamate is a major excitatory neurotransmitter that regulatesvirtually all activities of the CNS including motor control, learn-

ing and memory, and cognition. However, excess extracellularglutamate can result in neuronal dysfunction and death, a processknown as excitotoxicity (Lucas and Newhouse, 1957; Olney et al.,1974). Excess extracellular glutamate causes neuronal death by

Received March 17, 2017; revised Aug. 2, 2017; accepted Aug. 9, 2017.Author contributions: K.S., T.A., and K.T. designed research; K.S. and T.A. performed research; M.N., R.T., and

H.U.Z. contributed unpublished reagents/analytic tools; K.S., T.A., and K.T. analyzed data; K.S., T.A., and K.T. wrotethe paper.

This work was supported by the Strategic Research Program for Brain Sciences from the Ministry of Education,Culture, Sports, Science and Technology of Japan (to K.T.). Additional support was provided by a grant from MedicalResearch Institute (MRI), Tokyo Medical and Dental University (TMDU; to T.A.), a Grant-in-Aid for Japan Society for

the Promotion of Science Fellows (to K.S.), and Nanken-Kyoten, TMDU (to K.T.). We thank Dr. Masahiko Watanabe(Hokkaido University) for providing antibodies; Kei Watase (Center for Brain Integration Research, TMDU) for valu-able suggestions; and Shizuko Ichinose, Yuriko Sakamaki (Research Center for Medical and Dental Science, TMDU),Harumi Ishikubo, and Masako Hidaka (MRI, TMDU) for their technical support.

*K.S. and T.A. contributed equally to this work.The authors declare no competing financial interests.

Significance Statement

Despite glial glutamate transporter dysfunction leading to excitotoxicity has been documented in many neurological diseases, itremains unclear whether its dysfunction is a primary cause or secondary outcome of neuronal death at disease state. Here we showthe combined loss of glial glutamate transporters GLT1 and GLAST in spinal cord caused motor neuronal death and hindlimbparalysis. Further, our novel mutant exhibits the nuclear irregularities and calpain-mediated progressive nuclear pore complexdegradation. Our study reveals that glial glutamate transporter dysfunction is sufficient to cause motor neuronal death in vivo.

8830 • The Journal of Neuroscience, September 6, 2017 • 37(36):8830 – 8844

the disruption of intracellular organelles and aberrant proteolysisvia ion-sensitive protease activation that results from excessiveCa2� influx following the overstimulation of Ca2�-permeable glu-tamate receptors (Hollmann et al., 1991; Verdoorn et al., 1991; Banoet al., 2005; Kwak and Weiss, 2006; Lewerenz and Maher, 2015).Accumulating evidence implicates excitotoxicity involvement in thepathogenesis of many neurodegenerative diseases including Alzhei-mer’s disease, multiple sclerosis, Parkinson’s disease, Huntington’sdisease, frontotemporal dementia (FTD), and amyotrophic lateralsclerosis (ALS; Kawahara et al., 2004; Kwak and Weiss, 2006; Mehtaet al., 2013; Zhang et al., 2016), suggesting that these diseases mayshare excitotoxicity as a common pathogenic pathway. Thus, under-standing the molecular mechanisms underlying chronic excitotoxicneuronal death is of critical importance for the treatment of manyneurodegenerative diseases.

There may be a number of mechanisms by which excito-toxicity is initiated: increased glutamate level due to enhancedglutamate release; increased glutamate level due to glutamatetransporter dysfunction; and altered expression or sensitivity ofglutamate receptors. Glutamate transporter dysfunction is widelydocumented in many neurodegenerative diseases such as Alzhei-mer’s disease and ALS (Fontana, 2015; Miladinovic et al., 2015).There are five subtypes of Na�-dependent glutamate transport-ers, GLAST (EAAT1), GLT1 (EAAT2), EAAC1 (EAAT3), EAAT4,and EAAT5. GLAST and GLT1 are mainly expressed in astrocytes,while EAAT3, EAAT4, and EAAT5 are mainly expressed in neu-rons (Tanaka, 2000). Astroglial glutamate transporters (GLASTand GLT1) are the glutamate transporters mainly involved in themaintenance of physiological extracellular glutamate concentra-tions (Tanaka, 2005). In ALS, there is significant evidence tosuggest that the dysfunction of glial glutamate transporters playsan important role in the pathogenic pathways of AMPA receptor-mediated motor neuron death. ALS patients show reduced glu-tamate uptake in the motor cortex and spinal cord (Rothstein etal., 1990), which results from a reduction of the glial glutamatetransporter GLT1 (also known as EAAT2; Rothstein et al., 1995).In addition, recent studies have reported that the spinal cord oftransgenic rats expressing mutant human TDP-43 selectively inastrocytes exhibit a progressive deletion of GLAST, another glialglutamate transporter, as well as GLT1 (Tong et al., 2013). How-ever, it remains unknown whether the dysfunction of glial gluta-mate transporters is a primary cause or a secondary consequenceof motor neuron degeneration. To explore a causal role for glialglutamate transporter dysfunction in motor neuron death andthe signaling cascades leading to glutamate-mediated neuronaldeath, we generated a novel mouse model of chronic glutamateexcitotoxicity by deleting GLT1 and GLAST from the spinal cord,because mouse with a complete deficiency of GLT1 exhibitedseizure and premature death (Tanaka et al., 1997).

Materials and MethodsMice. All animal procedures were performed in accordance with theanimal experiment plan approved by the Tokyo Medical and DentalUniversity Animal Care and Use Committee. Mice were housed three tofive animals per cage and were maintained on a regular 12 h light/darkcycle (light period, 8:00 A.M. to 8:00 P.M.) at a constant 25°C. Food andwater were available ad libitum. In particular, when animals showedcomplete hindlimb paralysis, moistened food and water were made easily

accessible to the animals on the cage floor. In the rare presence of amoribund animal, the animal was killed. Animal experiments wereperformed according to the Internal Institutional Review Committee.Floxed-GLT1 mice, Hoxb8-Cre transgenic mice, and GLAST knock-out(KO) mice were previously described (Watase et al., 1998; Witschi et al.,2010; Cui et al., 2014; Aida et al., 2015). Both male and female mice wereused unless otherwise noted, and the number of male and female mice isdescribed in sample sizes by experiment. The age and number of miceused for all experiments are described in the figure legends.

Behavioral tests. Behavioral analyses were performed by investigatorsblind to the condition, genotype, and drug treatment groups.

Hanging wire test. Hanging wire tests were performed as previouslydescribed (Hanada et al., 2013). Briefly, male mice were placed on a wirenetting, and the netting was turned over and held 50 cm above the table-top for 60 s. The time until the hindlimbs disengaged from the wirenetting was recorded as the latency to fall.

Hindlimb reflex score. The posture of hindlimbs was scored as previ-ously described (Wils et al., 2010). Briefly, male mice were tail suspendedfor 14 s and scored using an established definition: 0, normal; 1, failure tostretch their hindlimbs; 2, hindlimb clasping; 3, hindlimb paralysis.

Histology. Histological analysis was performed as previously described(Cui et al., 2014; Aida et al., 2015). Mice deeply anesthetized with pento-barbital (100 mg/kg, i.p.) were fixed by perfusion with 4% paraformal-dehyde (PFA) in PBS. Brains and spinal cords were dissected and furtherfixed with 4% PFA overnight. Tissues were transferred to 30% sucrose/PBS for cryoprotection and separated into brain, cervical, thoracic, andlumbar spinal cords in OCT compound (Sakura). Cryosections wereprepared at a 20 �m thickness for spinal cords and a 50 �m thickness forbrain (CM3050s, Leica) and mounted on MAS-coated slides (Matu-nami). Cryosections were permeabilized and blocked with 0.3% TritonX-100, 1% bovine serum albumin (BSA), and 10% normal goat serum inPBS, and were incubated with primary antibodies overnight at 4°C. Forcholine acetyltransferase (ChAT), sections were heated in 10 mM citratebuffer, pH 6.0, at 100°C for 20 min then were permeabilized and blockedwith 0.3% Triton X-100, 1% BSA, and 10% normal horse serum in PBS.The following antibodies were used: polyclonal anti-GLT1 (1:5000; a giftfrom M. Watanabe, Hokkaido University, Sapporo, Japan; Yamada et al.,1998), polyclonal anti-ChAT (1:200; MAB144P, Merck Millipore), poly-clonal anti-glial fibrillary acidic protein (GFAP; 1:1000; Z0334, Dako),polyclonal anti-CD68 (1:100; MCA1957, AbD Serotec), monoclonalanti-NeuN (1:500; MAB377, Millipore), monoclonal anti-Nup153 (1:100; ab24700, Abcam), rabbit polyclonal anti-TDP-43 (1:100; 10782-2AP, Proteintech), goat polyclonal anti-Lamin B1 (1:200; sc-6217, SantaCruz Biotechnology), mouse monoclonal anti-KPNB1 (1:200; sc-137016, SantaCruz Biotechnology), polyclonal anti-RanBP1 (1:500; NB100-79814,Novus Biologicals), and polyclonal anti-cellular apoptosis susceptibility(CAS; 1:100; ab96755, Abcam) antibodies. Sections were washed, incu-bated with secondary antibodies conjugated with Alexa Fluor 488, 594, or633, and DAPI (Life Technologies) for 2 h, and mounted with Fluoro-mount (Diagnostic BioSystems). For ChAT and Lamin B1 staining, sec-tions were incubated with biotinylated donkey anti-goat IgG (1:200;Vector Laboratories) for 2 h, washed, and incubated with streptavidinconjugated with Alexa Fluor 488 (1:200; Invitrogen) for 2 h. For fluores-cent Nissl staining, sections were incubated with NeuroTrace 640/660Deep-Red Fluorescent Nissl Stain (1:100; Invitrogen) for 30 min, im-mersed in 0.3% Triton-X in PBS for 15 min, and washed two times inPBS. For GLT1 staining, sections were incubated with biotinylated goatanti-rabbit IgG (1:1000; Vector Laboratories) for 1 h, immersed in 1%H2O2/PBS, and incubated with AB complex (1:500; Vector Laboratories)for 1 h, and then visualized with a DAB substrate kit (Vector Laborato-ries). Images were acquired using an LSM710 Laser Scanning ConfocalMicroscope (Carl Zeiss) or an SZX16 Stereoscopic Microscope for GLT1immunostaining (Olympus).

Electron microscopy. Mice deeply anesthetized with pentobarbital(100 mg/kg, i.p.) were fixed by perfusion with 4% PFA and 2.5% glutar-aldehyde in 0.1 M phosphate buffer, pH 7.4. Spinal cords were dissectedand further fixed with same fixative overnight. After dehydration, sam-ples were embedded in Epon 812. Ultrathin sections were stained with

Correspondence should be addressed to Dr. Kohichi Tanaka, Laboratory of Molecular Neuroscience, MedicalResearch Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo, Tokyo 113-8510, Japan. E-mail:[email protected].

DOI:10.1523/JNEUROSCI.0730-17.2017Copyright © 2017 the authors 0270-6474/17/378831-15$15.00/0

Sugiyama, Aida et al. • Excitotoxic Motor Neuron Death In Vivo J. Neurosci., September 6, 2017 • 37(36):8830 – 8844 • 8831

2% uranyl acetate for 20 min and lead citrate solution for 5 min. Electronmicrographs were taken on an H7100 electron microscope (Hitachi).

Cell counts. Cell counts were performed using ImageJ software with thecell counter plug-in (National Institutes of Health, Bethesda, MD) byinvestigators who were blind to genotype and drug treatment. For motorneurons, the number of ChAT-positive cells in the bilateral ventral hornsof L3–L5 in three to nine serial sections was counted. For the otherneurons, NeuN-positive cells in laminae I–IV were counted. Laminaeboundaries were determined according to cytoarchitectonic criteria pre-viously described (Wootz et al., 2013). For glial cells, GFAP- and CD68-positive cells in bilateral ventral horns of L3–L5 in three serial sectionswere counted. Motor neurons showing nuclear irregularity were definedas motor neurons (large ventral horn neurons with a nuclear diameter of�10 �m) in which laminB1-immunolabeled nuclear contour showeddiscontinuous and frayed morphology, such as a ruffled edge and pedal-like shape (Freibaum et al., 2015). Nup153-negative motor neurons weredefined as motor neurons (large ventral horn neurons with a nucleardiameter of �10 �m) in which more than half of Nup153-immunolabeled nu-clear contours were lost. Motor neurons showing TDP-43 pathologywere defined as ChAT-positive cells displaying aberrant staining patternsof TDP-43, such as mislocalization to the cytoplasm (mislocalization),nuclear reduction without cytoplasmic immunoreactivity (reduction),and absence from both the nucleus and the cytoplasm (absence). CAS-negative motor neurons were defined as ChAT-positive cells in whichmore than half of CAS immunoreactivity was lost from the nucleus.

Western blot analysis. Western blot analysis was performed as previ-ously described (Aida et al., 2015). Brains and spinal cords were dissectedafter perfusion with ice-cold PBS. Brain hemisphere or lumbar spinalcords were homogenized in 0.32 M sucrose, 20 mM Tris-HCl, pH 7.5,1 mM NaVO4, 1 mM NaF, and a protease inhibitor cocktail (Roche). Afterthe calculation of protein concentrations by BCA assay (Sigma-Aldrich),samples were mixed with an equal amount of sample loading buffer (250 mM

Tris-HCl, pH 6.8, 4% SDS, 10% glycerol, and 1% �-mercaptoethanol) anddenatured by heating at 95°C for 10 min. Thirty micrograms of eachsample was separated by 10% SDS-PAGE, transferred to PVDF mem-branes (Merck Millipore), and blocked in Tris-buffered saline (TBS)with 0.5% Tween 20 (TBS-T) and 5% skim milk for 1 h at room temper-ature. The membranes were incubated with primary antibodies in TBS-Tcontaining 1% skim milk at 4°C overnight, washed, and incubated withhorseradish peroxidase (HRP)-conjugated secondary antibodies at roomtemperature for 1 h. The following antibodies were used: polyclonalanti-GLAST (1:1000; a gift from M. Watanabe, Hokkaido University),polyclonal anti-GLT1 (1:5000; a gift from M. Watanabe, Hokkaido Uni-versity), polyclonal anti-EAAC1 (1:500; sc-25658, Santa Cruz Biotech-nology), monoclonal anti-Nup62 (1:500; BD Biosciences), monoclonalanti-Nup153 (1:500; Abcam), polyclonal anti-Nup88 (1:1000; ab79785,Abcam), goat polyclonal anti-Lamin B1 (1:2000; sc-6217, Santa CruzBiotechnology), monoclonal anti-spectrin (1:5000; MAB1622, Milli-pore), rabbit polyclonal anti-p35 (1:250; sc-820, Santa Cruz Biotechnol-ogy), rabbit polyclonal anti-TDP-43 (1:2000; 10782-2AP, Proteintech),monoclonal anti-�-tubulin (1:5000; T7451; Sigma-Aldrich), monoclo-nal anti-�-actin (1:5000; sc-47778, Santa Cruz Biotechnology), andHRP-conjugated anti-rabbit or mouse IgG (1:5000; Jackson ImmunoRe-search) antibodies. The membranes were washed and visualized withLuminata Forte Western HRP substrate (Millipore). Gel images weretaken and quantified by measuring the ratio of the band intensities nor-malized to the intensity of �-actin or �-tubulin using Image Lab Software(Bio-Rad).

In vitro calpain activity analysis. In vitro calpain activity analysis waspreviously described (Sato et al., 2011). The mouse brains were homog-enized in 10 volumes (volume/weight) of HEPES buffer (20 mM HEPES,pH 7.5, 5 mM KCl, 2 mM MgCl2, 1 mM EGTA, and 1 mM dithiothreitol) at4°C using a Teflon pestle in a glass homogenizer. The homogenates werecentrifuged at 18,000 � g for 20 min, and the resulting supernatants werecollected. The supernatant was incubated in vitro with exogenous5 mM CaCl2 at 30°C. Proteolysis of Nup153, Nup88, Nup62, LaminB1,�-spectrin, and TDP-43 was assessed by immunoblotting, as describedabove.

Measurement of endogenous calpain activity. Calpain activity was mea-sured using a kit from BioVision following manufacturer instructions.Briefly, tissue lysates were prepared from lumbar ventral horn of eachmouse in extraction buffer and the homogenates were centrifuged at13,200 rpm for 5 min at 4°C. After collecting supernatant, proteinconcentration was determined by BCA assay (Sigma-Aldrich). Fifty mi-crograms of protein was used for the calpain assay, as described by themanufacturer. Fluorescence emitted from calpain substrates and a cleavedone was quantified using a fluorescence microplate reader equipped with a400 nm excitation filter, a 505 nm emission filter (Spark 10M, Tecan). Cal-pain activity was calculated as relative fluorescence units using Spark Con-trol Software (Tecan).

Drug treatment. Perampanel (Hanada et al., 2011) provided by Eisaiwas dissolved in 0.5% (w/v) methylcellulose (Wako Pure Chemicals) andorally administered to the mice at a dose of 20 mg/kg once a day from 6 to7 weeks of age. Memantine (Sigma-Aldrich) dissolved in saline was in-jected intraperitoneally into the mice at a dose of 10 mg/kg twice a dayfrom 6 to 7 weeks of age. MK-801 (Sigma-Aldrich) dissolved in saline wasinjected intraperitoneally into the mice at a dose of 3 mg/kg once a dayfrom 6 to 8 weeks of age. SNJ-1945 (Oka et al., 2006) provided by SenjuPharmaceutical Co. was dissolved in 0.5% (w/v) sodium carboxymethylcellulose (Wako Pure Chemicals) and orally administered to the mice ata dose of 200 mg/kg once a day from 6 to 7 weeks of age. KPT-350 (Haineset al., 2015) provided by Karyopharm Therapeutics was dissolved 0.6%Pluronic F-68 (w/v) and 0.6% PVP K-29/32 (w/v) in water and orallyadministered to the mice at a dose of 15 mg/kg every other day from 6 to7 weeks of age. The hanging wire test was performed 24 h after the finaladministration, and then the mice were fixed by perfusion with 4% PFA/PBS and subjected to quantification of motor neurons.

To examine the long-term effects of perampanel and SNJ-1945, micewere treated daily with perampanel (20 mg/kg, orally) or SNJ-1945(200 mg/kg, orally) for 42 d starting at 6 weeks of age. Since a 15 mg/kgdose of KPT-350 is not a safe dose for long-term treatment, we orallyadministered KPT-350 (7.5 mg/kg) three times per week from 6 to 12weeks of age (Haines et al., 2015). Drug-treated and vehicle-treated ani-mals were monitored daily for survival and weekly for grip strength. Inthe hanging wire test, we replaced the missing values in the latency to fallwith 0 s when mice died during the long-term treatment.

Mice were fixed 24 h after the final administration by perfusion with4% PFA/PBS and subjected to the quantification of motor neurons.

Experimental design. All experiments were performed with appropri-ate controls such as GLT1 flox/flox mice and vehicle-treated GLAST �/�/GLT1-cKO mice. The type, number, and sexes of the animals used foreach experiment are detailed in the figures as follows:

Figure 1C–F: 3 GLT1 flox/flox (control, 3 females), 3 Hoxb8-Cre/GLT1 flox/flox (3 females), 3 GLAST �/�/GLT1 flox/flox (3 females), and3 Hoxb8-Cre/GLT1 flox/flox/GLAST �/� (GLAST �/�/GLT1-cKO, 3 fe-males) mice.

Figure 1G–I: 15 control (15 males) and 16 GLAST �/�/GLT1-cKO(16 males) mice.

Figure 1J: 11 control (11 males) and 14 GLAST �/�/GLT1-cKO(14 males) mice.

Figure 2B: P5W, 4 control (4 males) and 5 GLAST �/�/GLT1-cKO (5males) mice; P6W, 4 control (4 males) and 4 GLAST �/�/GLT1-cKO (4males) mice; P7W, 3 control (3 males) and 4 GLAST �/�/GLT1-cKO(4 males) mice; P8W, 3 control (3 males) and 3 GLAST�/�/GLT1-cKO (3males) mice; P9W, 4 control (4 males) and 5 GLAST�/�/GLT1-cKO (5males) mice; and P20W, 3 control (1 male, 2 females) and 4 GLAST�/�/GLT1-cKO (1 male, 3 females) mice.

Figure 2D: P5W, 3 control (2 males, 1 female) and 3 GLAST �/�/GLT1-cKO (2 males, 1 female) mice; P7W, 4 control (4 males) and 4GLAST �/�/GLT1-cKO (4 males) mice; P9W, 3 control (3 males) and 4GLAST �/�/GLT1-cKO (4 males) mice; and P20W, 3 control (1 male, 2female) and 3 GLAST �/�/GLT1-cKO (1 male, 2 female) mice.

Figure 2 F, H: P5W, 3 control (2 males, 1 female) and 3 GLAST �/�/GLT1-cKO (2 males, 1 female) mice; P6W, 3 control (2 males, 1 female)and 3 GLAST �/�/GLT1-cKO (2 males, 1 female) mice; P7W, 4 control(4 males) and 4 GLAST �/�/GLT1-cKO (4 male) mice; P8W 3 control (2males, 1 female) and 3 GLAST �/�/GLT1-cKO (3 males); P9W, 3 control

8832 • J. Neurosci., September 6, 2017 • 37(36):8830 – 8844 Sugiyama, Aida et al. • Excitotoxic Motor Neuron Death In Vivo

(3 males) and 3 GLAST �/�/GLT1-cKO (3 males) mice; and P20W, 3control (1 male, 2 females) and 3 GLAST �/�/GLT1-cKO (1 male, 2females) mice.

Figure 3A: 15 vehicle-treated GLAST �/�/GLT1-cKO (15 males) and17 perampanel-treated GLAST �/�/GLT1-cKO (17 males) mice.

Figure 3D: 3 control (3 males), 4 vehicle-treated GLAST �/�/GLT1-cKO (4 males) and 5 perampanel-treated GLAST �/�/GLT1-cKO (5males) mice.

Figure 3E: 9 vehicle-treated GLAST �/�/GLT1-cKO (5 males, 4 fe-males) and 7 memantine-treated GLAST �/�/GLT1-cKO (4 males, 3 fe-males) mice.

Figure 3G: 3 vehicle-treated GLAST �/�/GLT1-cKO (3 females) and 4memantine-treated GLAST �/�/GLT1-cKO (1 male, 3 female) mice.

Figure 3H: 5 vehicle-treated GLAST �/�/GLT1-cKO (4 males, 1 fe-males) and 6 MK-801-treated GLAST �/�/GLT1-cKO (4 males, 2 fe-male) mice.

Figure 3J: 3 vehicle-treated GLAST �/�/GLT1-cKO (3 males) and 3MK-801-treated GLAST �/�/GLT1-cKO (2 male, 1 female) mice.

Figure 4C: P4W, 3 control (3 females) and 3 GLAST �/�/GLT1-cKO (3females) mice; P5W, 3 control (1 male, 2 females) and 4 GLAST �/�/GLT1-cKO (2 males, 2 females) mice; P6W, 4 control (4 males) and 6GLAST �/�/GLT1-cKO (3 males, 3 females) mice; P7W, 5 control (5males) and 4 GLAST �/�/GLT1-cKO (2 males, 2 females) mice; P8W, 3control (3 females) and 3 GLAST �/�/GLT1-cKO (2 male, 1 females)mice; and P9W, 3 control (2 males, 1 female) and 3 GLAST �/�/GLT1-cKO (2 males, 1 female) mice.

Figure 4E: P4W, 3 control (3 females) and 3 GLAST �/�/GLT1-cKO (3females) mice; P5W, 3 control (1 male, 2 females) and 4 GLAST �/�/GLT1-cKO (2 males, 2 females) mice; P6W, 3 control (1 male, 2 females)and 3 GLAST �/�/GLT1-cKO (1 male, 2 females) mice; and P7W, 3control (3 males) and 4 GLAST �/�/GLT1-cKO (4 males) mice.

Figure 4G: 4 control (3 males, 1 female) and 5 GLAST �/�/GLT1-cKO(4 males, 1 female) mice.

Figure 5C: P5W, 3 control (1 male, 2 females) and 3 GLAST �/�/GLT1-cKO (1 make, 2 females) mice; and P6W, 4 control (4 females) and3 GLAST �/�/GLT1-cKO (3 females) mice.

Figure 1. Conditional knockout of astroglial glutamate transporters in the spinal cord causes paralysis and motor neuron death. A, Schematic diagram showing the generation of GLAST �/�/GLT1-cKO mice. B, GLT1 immunohistochemistry in brain, cervival (C6), thoracic (T2 and T10), and lumbar (L5) spinal cord tissue at 3 months of age. Scale bars: top, 5 mm; bottom, 500 �m.C, D, Western blot analysis of GLT1 in the brains (C) and lumbar spinal cords (D) of control (GLT1 flox/flox), Hoxb8-Cre/GLT1 flox/flox, GLAST �/�/GLT1 flox/flox, and Hoxb8-Cre/GLT1 flox/flox /GLAST �/�

(GLAST �/�/GLT1-cKO) mice at 4 weeks of age (n � 3 for each groups). GLT1 monomer band intensities were normalized to �-actin. **p � 0.01. n.s., not significant ( post hoc Tukey-HSD test afterone-way ANOVA). E, Western blot analysis of GLAST in the lumbar spinal cord of control, Hoxb8-Cre/GLT1 flox/flox, GLAST �/�/GLT1 flox/flox, and GLAST �/�/GLT1-cKO mice at 4 weeks of age (n �3 for each groups). GLAST band intensities were normalized to �-actin. **p � 0.01 ( post hoc Tukey HSD test after one-way ANOVA). F, Western blot analysis of EAAC1 in the lumbar spinal cord ofcontrol, Hoxb8-Cre/GLT1 flox/flox, GLAST �/�/GLT1 flox/flox, and GLAST �/�/GLT1-cKO mice at 4 weeks of age (n � 3 for each groups). EAAC1 band intensities were normalized to �-actin. n.s., notsignificant (one-way ANOVA). G–I, Body weight changes ( G), hindlimb reflex score (H ), and hanging wire test (I ) of control (GLT1 flox/flox, n � 15) and GLAST �/�/GLT1-cKO animals (Hoxb8-Cre/GLT1 flox/flox /GLAST �/�, n � 16). ***p � 0.001 ( post hoc unpaired two-tailed t test at corresponding time-point after two-way repeated-measures ANOVA). J, Percentage survival of control(n � 11) and GLAST �/�/GLT1-cKO (n � 14) animals calculated using the Kaplan–Meier method ( p � 0.000016, log rank test). All data are expressed as the mean � SEM.


Figure 2. Loss of motor neurons and gliosis in GLAST �/�/GLT1-cKO mice. A, ChAT immunofluorescence of the lumbar ventral horn in control and GLAST �/�/GLT1-cKO mice at 5, 7, 9, and 20weeks of age. Scale bar, 50 �m. B, Quantification of ChAT-positive motor neurons from 5 to 9, and 20 weeks of age of control mice (P5W, n � 4; P6W, n � 4; P7W, n � 3; P8W, n � 3; P9W, n � 4;P20W, n � 3) and GLAST �/�/GLT1-cKO mice (P5W, n � 5; P6W, n � 4; P7W, n � 4; P8W, n � 3; P9W, n � 5; P20W, n � 4). *p � 0.05, **p � 0.05, ***p � 0.001. n.s., not significant (unpairedtwo-tailed t test). C, NeuN immunofluorescence in the lumbar dorsal horn from control and GLAST �/�/GLT1-cKO mice at 20 weeks of age. Scale bar, 200 �m. D, Quantification of NeuN-positiveneurons in the superficial dorsal horn (laminae I–IV) of the spinal cord at 5, 7, 9, and 20 weeks of control mice (P5W, n � 3; P7W, n � 4; P9W, n � 3; P20W, n � 3) and GLAST �/�/GLT1-cKO mice(P5W, n � 3; P7W, n � 4; P9W, n � 4; P20W, n � 3) mice. E, GFAP immunofluorescence in the lumbar ventral horn from control and GLAST �/�/GLT1-cKO mice at 5, 7, 9, and 20 weeksof age. Scale bar, 100 �m. F, Quantification of GFAP-positive cells in the lumbar ventral horn from 5 to 9, and 20 weeks of age of control mice (Figure legend continues.)


Figure 5D: P4W, 5 control (2 males, 3 females) and 6 GLAST �/�/GLT1-cKO (2 males, 4 females) mice; P5W, 6 control (3 males, 3 females)and 5 GLAST �/�/GLT1-cKO (3 males, 2 females) mice; and P6W, 5control (3 males, 2 females) and 6 GLAST �/�/GLT1-cKO (3 males, 3females) mice.

Figure 5F: P5W, 7 control (7 males) and 7 GLAST �/�/GLT1-cKO (7males) mice; P6W, 6 control (6 males) and 5 GLAST �/�/GLT1-cKO (3males, 2 females) mice; P7W, 5 control (5 males) and 6 GLAST �/�/GLT1-cKO (2 males, 4 females) mice; and P9W, 6 control (6 males) and5 GLAST �/�/GLT1-cKO (5 males) mice.

Figure 5H: 8 vehicle-treated GLAST �/�/GLT1-cKO (6 males, 2 fe-males) and 8 SNJ-1945-treated GLAST �/�/GLT1-cKO (7 males, 1 fe-males) mice.

Figure 5J: 5 vehicle-treated GLAST �/�/GLT1-cKO (5 males) and 6SNJ-1945-treated GLAST �/�/GLT1-cKO (6 males) mice.

Figure 5L: 4 vehicle-treated GLAST �/�/GLT1-cKO (4 males) and 4SNJ-1945-treated GLAST �/�/GLT1-cKO (4 males) mice.

Figure 5N: 4 vehicle-treated GLAST �/�/GLT1-cKO (4 males) and 5perampanel-treated GLAST �/�/GLT1-cKO (5 males) mice.

Figure 6C: 3 control (3 males) and 4 GLAST �/�/GLT1-cKO (3 males,1 female) mice.

Figure 6D: 10 vehicle-treated GLAST �/�/GLT1-cKO (5 males, 5 fe-males) and 9 KPT-350-treated GLAST �/�/GLT1-cKO (3 males, 6 fe-males) mice.

Figure 6G: 7 vehicle-treated GLAST �/�/GLT1-cKO (2 males, 5 fe-males) and 5 KPT-350-treated GLAST �/�/GLT1-cKO (1 male, 4 fe-males) mice.

Figure 6I: 3 vehicle-treated GLAST �/�/GLT1-cKO (1 male, 2 females)and 3 KPT-350-treated GLAST �/�/GLT1-cKO (3 females) mice.

Figure 7 A, B: 20 vehicle-treated GLAST �/�/GLT1-cKO (11 males, 9females) and 16 perampanel-treated GLAST �/�/GLT1-cKO (8 males, 8females) mice.

Figure 7D: 4 vehicle-treated GLAST �/�/GLT1-cKO (3 males, 1 fe-males) and 6 perampanel-treated GLAST �/�/GLT1-cKO (3 males, 3females) mice.

Figure 7 E, F: 18 vehicle-treated GLAST �/�/GLT1-cKO (9 males, 9females) and 16 SNJ-1945-treated GLAST �/�/GLT1-cKO (6 males, 10females) mice.

Figure 7H: 4 vehicle-treated GLAST �/�/GLT1-cKO (3 males, 1 fe-male) and 6 SNJ-1945-treated GLAST �/�/GLT1-cKO (2 males, 4 fe-males) mice.

Figure 7 I, J: 19 vehicle-treated GLAST �/�/GLT1-cKO (9 males, 10females) and 14 KPT-350-treated GLAST �/�/GLT1-cKO (7 males, 7females) mice.

Figure 7L: 4 vehicle-treated GLAST �/�/GLT1-cKO (2 males, 2 fe-males) and 6 KPT-350-treated GLAST �/�/GLT1-cKO (4 males, 2 fe-males) mice.

Statistical analysis. All data are expressed as the mean � SEM. Anunpaired two-tailed t test was used to compare differences between anytwo groups. One-way ANOVA with post hoc Tukey’s HSD test was usedto compare differences between among than three groups. For longitu-dinal observations, two-way repeated-measures ANOVA with post hocunpaired two-tailed t test at the corresponding time point was per-

4

(Figure legend continued.) (P5W, n � 3; P6W, n � 3; P7W, n � 4; P8W, n � 3; P9W, n � 3;P20W, n � 3) and GLAST �/�/GLT1-cKO mice (P5W, n � 3; P6W, n � 3; P7W, n � 4; P8W,n � 3; P9W, n � 3; P20W, n � 3) mice. *p � 0.05, **p � 0.01, ***p � 0.001. G, CD68immunofluorescence in the lumbar ventral horns from control and GLAST �/�/GLT1-cKO miceat 5, 7, 9, and 20 weeks of age. Scale bar, 100 �m. H, Quantification of CD68-positive cells in thelumbar ventral horn from 5 to 9, and 20 weeks of age of control mice (P5W, n � 3; P6W, n � 3;P7W, n � 4; P8W, n � 3; P9W, n � 3; P20W, n � 3) and GLAST �/�/GLT1-cKO mice (P5W,n � 3; P6W, n � 3; P7W, n � 4; P8W, n � 3; P9W, n � 3; P20W, n � 3). *p � 0.05. All dataare expressed as the mean � SEM. n.s., Not significant (unpaired two-tailed t test).

Figure 3. Overactivation of AMPA receptor but not NMDA receptor contributes to motor deficits and motor neuron loss in GLAST �/�/GLT1-cKO mice. A, Perampanel treatment delayed motordeficits in the hanging wire test (n � 15 for vehicle-treated and n � 17 for perampanel-treated GLAST �/�/GLT1-cKO mice). *p � 0.05 ( post hoc unpaired two-tailed t test at corresponding timepoint after two-way repeated-measures ANOVA). B, Amelioration of hindlimb paralysis in GLAST �/�/GLT1-cKO mice after perampanel treatment. Arrow indicates the fully paralyzed hindlimb ofvehicle-treated GLAST �/�/GLT1-cKO mice. Arrowhead indicates the hindlimb upon which the perampanel-treated GLAST �/�/GLT1-cKO mice could stand. C, ChAT immunofluorescence of thelumbar ventral horn from control, vehicle-treated, and perampanel-treated GLAST �/�/GLT1-cKO mice at 7 weeks of age. Scale bar, 100 �m. D, Quantification of ChAT-positive motor neurons at7 weeks of age (n � 3 for control mice, n � 4 for vehicle-treated mice, and n � 5 for perampanel-treated GLAST �/�/GLT1-cKO mice). *p � 0.05, ***p � 0.001 ( post hoc Tukey HSD test afterone-way ANOVA). E, Memantine treatment could not ameliorate motor deficits in the hanging wire test (n � 9 for vehicle-treated and n � 7 for memantine-treated GLAST �/�/GLT1-cKO mice).n.s., Not significant (two-way repeated-measures ANOVA). F, ChAT immunofluorescence of the lumbar ventral horn from vehicle-treated and memantine-treated GLAST �/�/GLT1-cKO mice at 7weeks of age. Scale bar, 100 �m. G, Quantification of ChAT-positive motor neurons at 7 weeks of age (n � 3 for vehicle-treated and 4 for memantine-treated GLAST �/�/GLT1-cKO mice). n.s., Notsignificant (unpaired two-tailed t test). H, MK-801 treatment could not ameliorate motor deficits in the hanging wire test (n � 5 for vehicle-treated and 6 for MK-801-treated GLAST �/�/GLT1-cKOmice). n.s., Not significant (two-way repeated-measures ANOVA). I ChAT immunofluorescence of the lumbar ventral horn from vehicle-treated and MK-801-treated GLAST �/�/GLT1-cKO mice at8 weeks of age. Scale bar, 100 �m. J, Quantification of ChAT-positive motor neurons at 8 weeks of age (n � 3 for each groups). n.s., Not significant (unpaired two-tailed t test). All data are expressedas the mean � SEM.


formed. Significant differences were analyzed by Kaplan-Meier and logrank tests for the survival rate. All statistical analyses were performedusing SPSS 24 (IBM). A p value of �0.05 was considered to be statisticallysignificant.

ResultsGeneration of spinal cord-specific astroglial glutamatetransporters KO mice (GLAST �/�/GLT1-cKO)Because astroglial glutamate transporters (GLT1 and GLAST) arecritical for the maintenance of extracellular glutamate and theprevention of chronic excitotoxicity (Tanaka et al., 1997; Wataseet al., 1998; Matsugami et al., 2006), we generated mice that

lacked GLT1 specifically in the spinal cord on a GLAST hetero-zygous background (Hoxb8-Cre/GLT1 flox/flox/GLAST�/� mice,hereafter, denoted as “GLAST�/�/GLT1-cKO mice”; Fig. 1A;Watase et al., 1998; Witschi et al., 2010; Cui et al., 2014; Aida et al.,2015). Immunohistochemistry and Western blot analysis ofGLT1 showed that the amount of GLT1 protein was graduallyreduced from the spinal cord caudal to the cervical level C6 andcompletely lost in the lumbar spinal cord in GLAST�/�/GLT1-cKO mice, which was consistent with the pattern of Cre-recombinase expression in the Hoxb8-Cre line [Fig. 1B–D; (Fig.1C: one-way ANOVA, F(3,8) � 0.11, p � 0.95; Fig. 1D: one-way

Figure 4. Nuclear contour irregularity and the loss of nuclear pore complex proteins are observed in spinal motor neurons of GLAST �/�/GLT1-cKO mice. A, Pseudocolored electron microscopicimages of motor neurons in lumbar ventral horns from control and GLAST �/�/GLT1-cKO mice at 6 weeks of age. The nucleus of the motor neuron is pseudocolored in light blue. Scale bar, 2 �m.B, Lamin B1 immunofluorescence of lumbar ventral horns from control and GLAST �/�/GLT1-cKO mice at 7 weeks of age. Sections were double labeled with anti-lamin B1 (green) and DAPI (blue).Rectangles in middle panel are enlarged in the right panel. Scale bars: left, 10 �m; right, 5 �m. C, Quantitative analysis of Lamin B1-immunostained sections. The percentage of large ventral hornneurons showing nuclear contour irregularity is higher in GLAST �/�/GLT1-cKO mice from 5 weeks of age. Large ventral horn neurons showing nuclear contour irregularity were counted from controlmice (P4W, n � 3; P5W, n � 3; P6W, n � 4; P7W, n � 5; P8W, n � 3; P9W, n � 3) and GLAST �/�/GLT1-cKO mice (P4W, n � 3; P5W, n � 4; P6W, n � 6; P7W, n � 4; P8W, n � 3; P9W, n �3). *p � 0.05, **p � 0.01, ***p � 0.001. n.s., Not significant (unpaired two-tailed t test). D, Nup153 immunofluorescence of lumbar ventral horns from control and GLAST �/�/GLT1-cKO miceat 6 weeks of age. Sections were triple labeled with anti-Nup153 (magenta), DAPI (blue), and Neurotrace (fluorescent Nissl stain, white). Rectangles in the middle panel are enlarged in right panel.Scale bars: left, 10 �m; right, 5 �m. E, Quantitative analysis of Nup153-immunostained sections. The percentage of Nup153-negative large ventral horn neurons is higher in GLAST �/�/GLT1-cKOmice from 6 weeks of age. Nup153-negative large ventral horn neurons were counted from control mice (n � 3 at each age), and GLAST �/�/GLT1-cKO mice (P4W, n � 3; P5W, n � 4; P6W, n �3; P7W, n � 4) mice. *p � 0.05, ***p � 0.001. n.s., Not significant (unpaired two-tailed t test). All data are expressed as the mean � SEM. F, Nup153 immunofluorescence of lumbar ChAT-positivemotor neurons from control and GLAST �/�/GLT1-cKO mice at 7 weeks of age. Sections were triple-labeled with anti-Nup153 (magenta), DAPI (blue), and ChAT (white). Rectangles in middle panelare enlarged in right panel. Scale bars: left, 10 �m; right, 5 �m. G, Quantitative analysis of Nup153(�)/ChAT(�) motor neurons. The percentage of Nup153 (�)/ ChAT(�) motor neurons is similarto that of Nup153(�) large ventral horn neurons in GLAST �/�/GLT1-cKO mice at 7 weeks of age (n � 4 for control and n � 5 for GLAST �/�/GLT1-cKO). **p � 0.01 (unpaired two-tailed t test).All data are expressed as the mean � SEM.


ANOVA, F(3,8) � 17.53, p � 0.00071; post hoc Tukey HSD tests,GLT1flox/flox vs Hoxb8-Cre/GLT1flox/flox, p � 0.0039; GLT1flox/flox vsGLAST�/�/GLT1flox/flox, p � 1.00; GLT1flox/flox vs GLAST�/�/GLT1-cKO,p�0.0044;Hoxb8-Cre/GLT1flox/flox vsGLAST�/�/GLT1flox/flox,p � 0.0036; Hoxb8-Cre/GLT1flox/flox vs GLAST�/�/GLT1-cKO, p �

1.00; GLAST�/�/GLT1 flox/flox vs GLAST�/�/GLT1-cKO, p �0.0041)]. Western blot analysis of GLAST revealed an 50% re-duction in the spinal cords of GLAST�/�/GLT1 flox/flox andGLAST�/�/GLT1-cKO mice when compared with GLT1 flox/flox

(hereafter denoted as “control”) and Hoxb8-Cre/GLT1flox/flox

Figure 5. Calpain overactivation contributes to motor deficits and motor neuron loss in GLAST �/�/GLT1-cKO mice. A, Representative immunoblot that shows proteolysis of Nup153, Nup88,Nup62, laminB1, and �-spectrin in mouse brain homogenate that was incubated with exogenous calcium and/or a calpain inhibitor, SNJ-1945 (100 �M). B, Representative immunoblot showingproteolysis of p35 in lumbar ventral horn from control and GLAST �/�/GLT1-cKO mice at 5 and 6 weeks of age. C, Quantification of the p25/p35 ratio from control mice (P5W, n � 3; P6W, n � 4)and GLAST �/�/GLT1-cKO mice (n � 3 at each age). Band intensities of p35 and p25 were normalized to �-tubulin. **p � 0.01. n.s., Not significant (unpaired two-tailed t test). D, Measurementof calpain activity of lumbar ventral horn form control mice (P4W, n � 5; P5W, n � 6; P6W, n � 5) and GLAST �/�/GLT1-cKO mice (P4W, n � 6; P5W, n � 5; P6W, n � 6). Relative fluorescenceunits (RFUs) were compared by calculating the fold difference in enzyme activity. *p � 0.05; n.s., Not significant (unpaired two-tailed t test). E, TDP-43 immunofluorescence in the lumbar ventralhorns from control at 7 weeks of age (the top row of a panel) and GLAST �/�/GLT1-cKO mice at 7 (the second and third rows of a panel) and 9 (the bottom row of a panel) weeks of age. Sections weredouble labeled with TDP-43 (red) and ChAT (white). Nuclei were visualized with DAPI. The second row of a panel shows mislocalization of TDP-43 to the cytoplasm in motor neurons ofGLAST �/�/GLT1-cKO mice. The third row of a panel shows reduction of nuclear TDP-43 without cytoplasmic TDP-43 immunoreactivity in motor neurons of GLAST �/�/GLT1-cKO mice. The bottomrow of a panel shows loss of TDP-43 from both the nucleus (yellow arrowhead) and the cytoplasm in motor neurons of GLAST �/�/GLT1-cKO mice. Scale bar, 10 �m. F, The percentage of motorneurons showing aberrant staining pattern of TDP-43 from 5, 6, 7, and 9 weeks of age of control mice (P5W, n � 7; P6W, n � 6; P7W, n � 5; P9W, n � 6) and GLAST �/�/GLT1-cKO mice (P5W,n � 7; P6W, n � 5; P7W, n � 6; P9W, n � 5). *p � 0.05, **p � 0.01 (unpaired two-tailed t test). G, Representative immunoblot that shows proteolysis of TDP-43 and �-actin in mouse brainhomogenate that was incubated with exogenous calcium and/or a calpain inhibitor, SNJ-1945 (100 �M). Black and white arrowheads indicate a full-length and cleaved fragment of TDP-43,respectively. H, SNJ-1945 treatment delayed motor deficits in the hanging wire test (n � 8 for each groups). *p � 0.05 ( post hoc unpaired two-tailed t test at corresponding time point aftertwo-way repeated-measures ANOVA). I, ChAT immunofluorescence of the lumbar ventral horns from vehicle-treated and SNJ-1945-treated GLAST �/�/GLT1-cKO mice at 7 weeks of age. Scale bar,100 �m. J, Quantification of ChAT-positive motor neurons at 7 weeks of age (n � 5 for vehicle-treated and n � 6 for SNJ-1945-treated GLAST �/�/GLT1-cKO mice). **p � 0.01 (unpairedtwo-tailed t test). K, Nup153 immunofluorescence of lumbar ventral horns from vehicle-treated and SNJ-1945-treated GLAST �/�/GLT1-cKO mice at 7 weeks of age. Sections were triple labeledwith anti-Nup153 (magenta), DAPI (blue), and Neurotrace (fluorescent Nissl stain, white). Rectangles in the middle panel are enlarged in the right panel. Scale bars: left, 10 �m; right, 5 �m.L, Quantitative analysis of Nup153-immunostained sections. The percentage of Nup153-negative large ventral horn neurons decreased in GLAST �/�/GLT1-cKO mice treated with SNJ-1945 (n � 4 for eachgroups). **p�0.01 (unpaired two-tailed t test). M, Nup153 immunofluorescence of lumbar ventral horns from vehicle-treated and perampanel-treated GLAST �/�/GLT1-cKO mice at 7 weeks of age. Sectionswere triple labeled with anti-Nup153 (magenta), DAPI (blue), and Neurotrace (fluorescent Nissl stain, white). Rectangles in the middle panel are enlarged in the right panel. Scale bars: left, 20�m; right, 5�m.N, Quantitative analysis of Nup153-immunostained sections. The percentage of Nup153-negative large ventral horn neurons decreased in GLAST �/�/GLT1-cKO mice treated with perampanel (n � 4 forvehicle-treated and n � 5 for perampanel-treated GLAST �/�/GLT1-cKO mice). *p � 0.05 (unpaired two-tailed t test). All data are expressed as the mean � SEM.


mice (Fig. 1E [one-way ANOVA, F(3,8) � 22.81, p � 0.00028; posthoc Tukey HSD tests, control vs Hoxb8-Cre/GLT1flox/flox, p � 0.95;control vs GLAST�/�/GLT1flox/flox, p � 0.0027; control vsGLAST�/�/GLT1-cKO, p � 0.0020; Hoxb8-Cre/GLT1 flox/flox vsGLAST�/�/GLT1 flox/flox, p � 0.0015; Hoxb8-Cre/GLT1 flox/flox

vs GLAST�/�/GLT1-cKO, p � 0.0012; GLAST�/�/GLT1 flox/flox

vs GLAST�/�/GLT1-cKO, p � 0.99]). There was no compensa-tory upregulation of other glutamate transporters (GLAST andEAAC1) in Hoxb8-Cre/GLT1flox/flox, GLAST�/�/GLT1 flox/flox,and GLAST�/�/GLT1-cKO mice (Fig. 1E,F [Fig. 1F: one-wayANOVA, F(3,8) � 0.095, p � 0.96]). Thus, we succeeded in gen-erating spinal cord-specific astroglial glutamate transportersknock-out mice.

Impaired motor function and motor neuron loss inGLAST �/�/GLT1-cKO miceThe most appropriate controls for GLAST�/�/GLT1-cKO miceare GLAST�/�/GLT1 flox/flox mice. However, there was no differ-

ence in the expression levels of GLAST and GLT1 betweenGLAST �/�/GLT1 flox/flox and GLAST �/� mice. In addition,GLAST �/� mice did not show apparent survival differences orany obvious abnormalities in basic motor and neurological func-tion compared with wild-type mice (Watase et al., 1998; Karlssonet al., 2009; Aida et al., 2015). Thus, we used GLT1 flox/flox mice ascontrols instead of GLAST�/�/GLT1 flox/flox mice in this study.GLAST�/�/GLT1-cKO mice had lower body weights than didthe controls starting at 7 weeks of age [Fig. 1G (two-way repeated-measures ANOVA for genotype � age interaction: F(4,116) �41.40, p � 1.8E-21; post hoc unpaired two-tailed t tests: P5W,t(29) � 0.41, p � 0.69; P6W, t(29) � 1.10, p � 0.28; P7W, t(29) �3.89, p � 0.00054; P8W, t(29) � 5.42, p � 7.8E-06; P9W, t(29) �8.46, p � 2.6E-09)]. They displayed an impaired hindlimb reflexby 7 weeks of age, after which they progressively lost grip strengthand became fully paralyzed in the hindlimbs by 8 weeks of age[Fig. 1H, I (Fig. 1H: two-way repeated-measures ANOVA for ge-notype � age interaction, F(4,116) � 62.03, p � 6.2E-28; post hoc

Figure 6. Nuclear export inhibitor KPT-350 transiently ameliorates motor deficits, motor neuron loss in GLAST �/�/GLT1-cKO mice. A, KPNB1 immunofluorescence of motor neurons in thelumbar ventral horn from control and GLAST �/�/GLT1-cKO mice at 7 weeks of age. Sections were triple labeled with anti-KPNB1 (green), DAPI (blue), and anti-ChAT (white). Scale bar, 10 �m.B, RanBP-1 immunofluorescence of motor neurons in the lumbar ventral horn from control and GLAST �/�/GLT1-cKO mice at 7 weeks of age. Sections were triple labeled with anti-RanBP-1 (green),DAPI (blue), and anti-ChAT (white). Scale bar, 10 �m. C, CAS immunofluorescence of motor neurons in the lumbar ventral horn from control and GLAST �/�/GLT1-cKO mice at 7 weeks of age.Sections were triple labeled with anti-CAS (red), DAPI (blue), and anti-ChAT (white). Arrowheads indicate the reduction or absence of nuclear CAS immunoreactivity in GLAST �/�/GLT1-cKO mice.Scale bar, 10 �m. D, KPT-350 treatment delayed motor deficits in the hanging wire test (n � 10 for vehicle-treated and n � 9 for KPT-350-treated GLAST �/�/GLT1-cKO mice). *p � 0.05 ( posthoc unpaired two-tailed t test at corresponding time point after two-way repeated-measures ANOVA). E, Amelioration of hindlimb paralysis in GLAST �/�/GLT1-cKO mice after KPT-350 treatment.Arrow indicates the fully paralyzed hindlimb of the vehicle-treated GLAST �/�/GLT1-cKO mice. Arrowhead indicates the hindlimb upon which the KPT-350-treated GLAST �/�/GLT1-cKO micecould stand. F, ChAT immunofluorescence of the lumbar ventral horn from vehicle-treated and KPT-350-treated GLAST �/�/GLT1-cKO mice at 7 weeks of age. Scale bar, 100 �m. G, Quantificationof ChAT-positive motor neurons at 7 weeks of age (n � 7 for vehicle-treated and n � 5 for KPT-350-treated GLAST �/�/GLT1-cKO mice). ***p � 0.001 (unpaired two-tailed t test). H, Nup153immunofluorescence of lumbar ventral horns from vehicle-treated and KPT-350-treated GLAST �/�/GLT1-cKO mice at 7 weeks of age. Sections were triple labeled with anti-Nup153 (magenta),DAPI (blue), and Neurotrace (fluorescent Nissl stain, white). Rectangles in the middle panel are enlarged in the right panel. Scale bars: left, 20 �m; right, 5 �m. (I) Quantitative analysis ofNup153-immunostained sections. The percentage of Nup153-negative large ventral horn neurons is not changed in GLAST �/�/GLT1-cKO mice treated with KPT-350 (n � 3 for each groups). n.s.,Not significant (unpaired two-tailed t test). All data are expressed as the mean � SEM.


unpaired two-tailed t tests, P5W, t(29) � 0.98, p � 0.33; P6W, t(29) �1.14, p � 0.26; P7W, t(29) � 3.79, p � 0.0007; P8W, t(29) � 13.75,p � 3.1E-14; P9W, t(29) � 13.97, p � 2.1E-14; Fig. 1I: two-wayrepeated-measures ANOVA for genotype � age interaction,F(4,116) � 37.49, p � 4.2E-20; post hoc unpaired two-tailed t tests,P5W, t(29) � 1.01, p � 0.32; P6W, t(29) � 0.16, p � 0.88; P7W, t(29) �1.89, p � 0.072; P8W, t(29) � 8.03, p � 7.4E-09; P9W, t(29) � 8.80,p � 1.1E-09)]. Thereafter, they had shortened life spans [Fig. 1J(log rank test, p � 0.000016)]. There was no significant differencein progression of motor deficits and survival rate between male andfemale GLAST�/�/GLT1-cKO mice (data not shown). In someGLAST�/�/GLT1-cKO mice, spasms of upper and lower limb mus-cles were observed, which were followed by an extremely rapid dis-ease progression with mice dying within 3–4 d.

Because GLAST�/�/GLT1-cKO mice showed progressivemotor deficits that resulted in hindlimb paralysis, we next exam-

ined whether GLT1 and GLAST deletion caused motor neuronloss and gliosis. ChAT immunolabeling showed motor neuronloss in lumbar ventral horn of GLAST�/�/GLT1-cKO mice be-ginning at 7 weeks of age with a peak at 8 weeks of age [Fig. 2A,B(Fig. 2B: unpaired two-tailed t tests, P5W, t(7) � 0.98, p � 0.36;P6W, t(6) � 0.38, p � 0.72; P7W, t(5) � 1.78, p � 0.14; P8W, t(4) �3.70, p � 0.021; P9W, t(7) � 4.75, p � 0.0021; P20W, t(5) � 10.85,p � 0.00012). In contrast, there was no change in the numberof NeuN-positive neurons in the superficial dorsal horn inGLAST�/�/GLT1-cKO mice [Fig. 2C,D (Fig. 2D: unpaired two-tailed t tests, P5W, t(4) � 0.26, p � 0.81; P7W, t(6) � 0.73, p �0.49; P9W, t(5) � 0.20, p � 0.85; P20W, t(4) � 0.06, p � 0.96)].Furthermore, enhanced immunoreactivity for GFAP, a marker ofastroglia, and CD68, a marker of activated microglia, was de-tected in the ventral horn of the spinal cord of GLAST�/�/GLT1-cKO mice beginning at 7 weeks of age (symptomatic stage; Fig.

Figure 7. Effects of long-term treatment with perampanel, SNJ-1945, and KPT-350 in GLAST �/�/GLT1-cKO mice. A, Perampanel treatment for 6 weeks delayed motor deficits in the hangingwire test (n � 20 for vehicle-treated and n � 16 for perampanel-treated GLAST �/�/GLT1-cKO mice). B, Percentage survival of vehicle-treated (n � 20) and perampanel-treated (n � 16)GLAST �/�/GLT1-cKO mice calculated using the Kaplan–Meier method ( p � 0.076, log rank test). n.s., Not significant. C, ChAT immunofluorescence of the lumbar ventral horn from vehicle-treatedand perampanel-treated GLAST �/�/GLT1-cKO mice at 12 weeks of age. D, Quantification of ChAT-positive motor neurons at 12 weeks of age (n � 4 for vehicle-treated and n � 6 forperampanel-treated GLAST �/�/GLT1-cKO mice). E, SNJ-1945 treatment for 6 weeks delayed motor deficits in the hanging wire test (n � 18 for vehicle-treated and n � 16 for SNJ-1945-treatedGLAST �/�/GLT1-cKO mice). F, Percentage survival of vehicle-treated (n � 18) and SNJ-1945-treated (n � 16) GLAST �/�/GLT1-cKO mice calculated using the Kaplan–Meier method ( p � 0.026,log rank test). *p � 0.05. G, ChAT immunofluorescence of the lumbar ventral horn from vehicle-treated and perampanel-treated GLAST �/�/GLT1-cKO mice at 12 weeks of age. H, Quantificationof ChAT-positive motor neurons at 12 weeks of age (n � 4 for vehicle-treated and n � 6 for SNJ-1945-treated GLAST �/�/GLT1-cKO mice). I, Amelioration of motor dysfunction by KPT-350treatment was observed at 7 weeks of age but not at later (n � 19 for vehicle-treated and n � 14 for KPT-350-treated GLAST �/�/GLT1-cKO mice). J, Percentage survival of vehicle-treated (n �19) and KPT-350-treated (n � 14) GLAST �/�/GLT1-cKO mice calculated using the Kaplan-Meier method ( p � 0.86, logrank test). n.s., Not significant. K, ChAT immunofluorescence of the lumbarventral horn from vehicle-treated and KPT-350-treated GLAST �/�/GLT1-cKO mice at 12 weeks of age. L, Quantification of ChAT-positive motor neurons at 12 weeks of age (n�4 for vehicle-treatedand n � 6 for KPT-350-treated GLAST �/�/GLT1-cKO mice). Scale bars: C, G, K, 100 �m. A, E, I, *p � 0.05, **p � 0.01, ***p � 0.001 ( post hoc unpaired two-tailed t test at corresponding timepoint after two-way repeated-measures ANOVA). D, H, L, *p � 0.05. n.s., Not significant (unpaired two-tailed t test). Red broken lines represent the first day of administration. All data are expressedas the mean � SEM.


2E,G). Quantification indicated a significant increase of GFAP-positive and CD68-positive cells in the ventral horn of the spinalcord of GLAST�/�/GLT1-cKO mice beginning at 8 weeks of age[Fig. 2F,H [Fig. 2F: unpaired two-tailed t tests, P5W, t(4) � 0.33,p � 0.76; P6W, t(4) � 0.86, p � 0.44; P7W, t(6) � 0.55, p � 0.60;P8W, t(4) � 8.39, p � 0.0011; P9W, t(4) � 19.97, p � 3.7E-05;P20W, t(4) � 4.51, p � 0.011; Fig. 2H: P5W, t(4) � 1.09, p �0.34; P6W, t(4) � 1.34, p � 0.25; P7W, t(6) � 1.39, p � 0.21; P8W,t(4) � 2.91, p � 0.044; P9W, t(4) � 4.10, p � 0.015; P20W, t(4) �4.00, p � 0.016)].

Together, these results suggest that the dysfunction of astro-glial glutamate transporters, GLT1 and GLAST, induced spinalmotor neuron loss, resulting in severe hindlimb paralysis in vivo.

Involvement of AMPA receptors overactivation in spinalmotor neuron loss of GLAST �/�/GLT1-cKO miceExcess extracellular glutamate due to the dysfunction of astroglialglutamate transporters induces neuronal death via overactivationof ionotropic glutamate receptors. In particular, previous workshave demonstrated the contribution of AMPA receptors (Coura-tier et al., 1993; Carriedo et al., 1996; Kwak and Weiss, 2006;Hideyama et al., 2010; Akamatsu et al., 2016; Yamashita et al.,2017b) and/or NMDA receptors to spinal motor neuron deaths(Mitchell et al., 2010; Sasabe et al., 2007, 2012). To determine thesubtype of ionotropic glutamate receptor that is involved in mo-tor neuron death in GLAST�/�/GLT1-cKO mice, we examinedthe effects of AMPA and NMDA receptor antagonists. We treatedGLAST�/�/GLT1-cKO mice with perampanel (Hanada et al.,2011), an AMPA receptor antagonist that is used as an antiepi-leptic drug. Akamatsu et al. (2016) reported that perampanel (20mg/kg) prevented the progression of the ALS phenotype in theAR2 mice, in which an RNA editing enzyme adenosine deami-nase acting on RNA 2 (ADAR2) is conditionally knocked out inthe motor neurons. Thus, GLAST�/�/GLT1-cKO mice weretreated daily with perampanel (20 mg/kg, orally) starting at 6weeks of age. Perampanel administration for 7 d prevented motordeficits and motor neuron loss in GLAST�/�/GLT1-cKO mice[Fig. 3A–D (Fig. 3A: two-way repeated-measures ANOVA fordrug � time interaction, F(1,30) � 4.76, p � 0.037; post hoc un-paired two-tailed t test, P7W, t(30) � 2.48, p � 0.019; Fig. 3D:one-way ANOVA, F(2,9) � 18.93, p � 0.0006; post hoc Tukey’sHSD tests, control vs vehicle-treated GLAST�/�/GLT1-cKO,p � 0.00048; control vs perampanel-treated GLAST�/�/GLT1-cKO, p � 0.045; vehicle-treated vs perampanel-treated GLAST�/�/GLT1-cKO, p � 0.0104)]. Next, to investigate the role of NMDAreceptors in spinal motor neuron death in GLAST�/�/GLT1-cKO mice, we treated GLAST�/�/GLT1-cKO mice with meman-tine (Lipton, 2006), an NMDA receptor antagonist. Wang andZhang (2005) reported that treating SOD1 (G93A) mice withmemantine (10 mg/kg) significantly delayed the disease progres-sion and prolonged survival. We administered memantine (10mg/kg, i.p., twice per day) every day for 7 d to GLAST�/�/GLT1-cKO mice at 6 weeks of age. Memantine treatment for 7 d had noeffect on motor function decline and motor neuron loss [Fig.3E–G (Fig. 3E: two-way repeated-measures ANOVA for drug �time interaction, F(1,14) � 1.71, p � 0.21; Fig. 3G: unpaired two-tailed t test, t(5) � 1.25, p � 0.27)]. Furthermore, we examined theeffect of another NMDA receptor antagonist, MK-801. Gill et al.(1987) demonstrated that systemic administration of MK-801(3 mg/kg) completely protected ischemia-induced hippocampaldamage in the gerbil. We administered MK-801 (3 mg/kg, i.p.)every day for 14 d to GLAST�/�/GLT1-cKO mice at 6 weeks ofage. MK-801 treatment for 14 d could not prevent the progres-

sion of motor deficits and motor neuron loss [Fig. 3H–J (Fig. 3H:two-way repeated-measures ANOVA for drug � time interac-tion, F(2,18) � 0.02, p � 0.98; Fig. 3J: unpaired two-tailed t test,t(4) � 0.83, p � 0.46)].

These results suggest that overstimulation of AMPA recep-tors, but not NMDA receptors, induces motor neuron loss andsevere motor deficits in this new in vivo model of chronic spinalcord excitotoxicity.

Calpain-dependent degradation of nuclear pore complexproteins in GLAST �/�/GLT1-cKO miceWe next examined the mechanisms by which the overactivationof AMPA receptors results in motor neuron death. An electronmicroscopic analysis revealed nuclear irregularities in spinal mo-tor neurons of GLAST�/�/GLT1-cKO mice (Fig. 4A), which re-sembles the pathologies of ALS and Parkinson’s disease patients(Kinoshita et al., 2009; Liu et al., 2012). The nuclear irregularityseen by electron microscopy was accentuated by lamin B1 immu-nostaining. In the large ventral horn neurons of control mice witha nuclear diameter of �10 �m, lamin B1 immunoreactivity wasdistributed predominantly as smooth nuclear contours (Fig. 4B).In contrast, the percentage of large ventral horn neurons exhib-iting nuclear contour irregularity such as flayed and discontinu-ous morphology is significantly higher in GLAST�/�/GLT1-cKOmice as early as 5 weeks of age (presymptomatic stage; Fig. 4B,C).The large ventral horn neurons with an irregular nuclear contourbecame the most common type of large ventral horn neurons inGLAST�/�/GLT1-cKO mice from 7 week of age to 9 weeks of age[symptomatic stage; Fig. 4C (unpaired two-tailed t tests, P4W,t(4) � 0.33, p � 0.76; P5W, t(5) � 3.72, p � 0.014; P6W, t(8) �4.38, p � 0.0024; P7W, t(7) � 7.39, p � 0.00015; P8W, t(4) � 5.16,p � 0.0067; P9W, t(4) � 6.60, p � 0.0027)]. These observationsprompted an examination of nuclear pore complexes (NPCs)that were embedded in the nuclear envelope in the large ventralhorn neurons of GLAST�/�/GLT1-cKO mice. We examined theimmunohistochemical distribution of the major protein compo-nent of NPCs, Nup153 (Fahrenkrog and Aebi, 2003; Kinoshita etal., 2009; Freibaum et al., 2015). Whereas Nup153 immunoreac-tivity was distributed predominantly along smooth nuclear con-tours in controls, it was reduced in large ventral horn neurons ofGLAST�/�/GLT1-cKO mice at 6 weeks of age [Fig. 4D,E (Fig.4E: unpaired two-tailed t tests, P4W, t(4) � 1.48, p � 0.21; P5W,t(5) � 1.30, p � 0.25; P6W, t(4) � 3.27, p � 0.031; P7W, t(5) �10.68, p � 0.00012)]. Double immunostaining of Nup153 andChAT revealed that the percentage of Nup 153-negative motorneurons is significantly increased to the same extent as Nup 153-negative large ventral horn neurons in GLAST�/�/GLT1-cKOmice at 7 weeks of age [Fig. 4F,G (Fig. 4G: unpaired two-tailed ttest, t(7) � 5.12, p � 0.0014)]. These results indicate the reducedNPC expression was occurred in the spinal motor neurons ofGLAST�/�/GLT1-cKO mice.

The loss of NPC protein immunolabeling on the nuclear en-velope suggests NPC protein degradation. Recent studies showedthat nucleoporins were cleaved by Ca 2�-activated protease cal-pain (Bano et al., 2010; Yamashita et al., 2017a). Consistent withthis, an in vitro assay (Sato et al., 2011) using mouse brain ho-mogenates displayed that the amounts of detectable full-lengthNup62, Nup88, and Nup153 were decreased in a Ca 2�-depen-dent manner, whereas SNJ-1945 (Oka et al., 2006), a calpaininhibitor, reversed the loss of these proteins (Fig. 5A). However,laminB1 was not a calpain substrate (Fig. 5A). In addition, weobserved that the cleavage of p35, which is well known as a cal-pain substrate (Lee et al., 2000), was significantly enhanced in the


spinal cords of GLAST�/�/GLT1-cKO mice at 6 weeks of agecompared with the control mice [Fig. 5B,C (Fig. 5C: unpairedtwo-tailed t tests, P5W, t(4) � 0.97, p � 0.39; P6W, t(5) � 5.10, p �0.0038)]. These results suggest that calpain is hyperactivated inthe spinal cord of GLAST�/�/GLT1-cKO mice. To confirm this,we measured calpain activity of the spinal cords. An increase incalpain activity in lumbar ventral horn was observed in GLAST�/�/GLT1-cKO mice as early as 5 weeks of age [Fig. 5D (unpairedtwo-tailed t tests, P4W, t(9) � 0.28, p � 0.79; P5W, t(9) � 3.05, p �0.014; P6W, t(9) � 2.90, p � 0.018)]. Yamashita et al. (2012,2017b) and Yamashita and Kwak (2014) have reported that TARDNA binding protein (TDP-43), one of the disease-related pro-teins of ALS and FTD, was also cleaved by calpain (Akamatsu etal., 2016). Thus, we assessed the immunohistochemical analysisfor TDP-43. Immunofluorescence for TDP-43 demonstratedthat TDP-43 localized exclusively to the nuclei in all of the spinalmotor neurons in control mice, whereas some spinal motor neu-rons in GLAST�/�/GLT1-cKO mice exhibited aberrant stainingpatterns of TDP-43, such as mislocalization to the cytoplasm(mislocalization), nuclear reduction without cytoplasmic immu-noreactivity (reduction), and the absence from both the nucleusand the cytoplasm (absence; Fig. 5E). Although the number ofmotor neurons showing aberrant staining patterns of TDP-43was very low, we detected such motor neurons in GLAST�/�/GLT1-cKO mice as early as 5 weeks of age [Fig. 5F (unpairedtwo-tailed t tests, P5W, t(12) � 3.32, p � 0.0061; P6W, t(9) � 2.84,p � 0.019; P7W, t(9) � 3.94, p � 0.0034; P9W, t(9) � 2.53, p �0.032)]. The percentage of motor neurons devoid of TDP-43increased between 7 and 9 weeks of age when motor neuron losswas prominent (Fig. 5F). Consistent with previous reports (Ya-mashita et al., 2012, 2017b; Akamatsu et al., 2016), TDP-43 wascleaved by calpain (Fig. 5G), suggesting that various immuno-staining patterns of TDP-43 in motor neurons of GLAST�/�/GLT1-cKO mice may result from cleavage by calpain.

To verify whether calpain-mediated NPC degradation waslinked to motor deficits and neuronal death of GLAST�/�/GLT1-cKO mice, we examined the effects of a blood– brainbarrier-permeable calpain inhibitor, SNJ-1945, in GLAST�/�/GLT1-cKO mice. Imai et al. (2010) reported that SNJ-1945 (200mg/kg) significantly prevented light-induced photoreceptor de-generation in the mice. Thus, we treated mice daily with SNJ-1945 (200 mg/kg, orally) starting at 6 weeks of age. One week ofSNJ-1945 treatment prevented the loss of grip strength of thehindlimbs in GLAST�/�/GLT1-cKO mice [Fig. 5H (two-wayrepeated-measures ANOVA for drug � time interaction, F(1,14) �8.59, p � 0.011; post hoc unpaired two-tailed t test, P7W, t(14) �2.93, p � 0.011)]. Furthermore, SNJ-1945 delayed the loss ofspinal motor neurons and Nup153 immunoreactivity [Fig. 5I–L(Fig. 5J: unpaired two-tailed t test, t(9) � 3.30, p � 0.0093; Fig. 5L:unpaired two-tailed t test, t(6) � 4.86, p � 0.0028)]. In addition,we also investigated a role for AMPA receptors in the degradationof Nup153 in GLAST�/�/GLT1-cKO mice. One week of peram-panel treatment (20 mg/kg, orally) prevented the degradation ofNup153 [Fig. 5M,N (Fig. 5N: unpaired two tailed t test, t(7) �3.34, p � 0.012)] as well as the death of spinal motor neurons(Fig. 3C,D) in GLAST�/�/GLT1-cKO mice.

Together, these findings suggest that the hyperactivation ofcalpain due to the overactivation of AMPA receptors is involvedin the degradation of the major protein components of NPCs,which is associated with excitotoxic spinal motor neuron death inGLAST�/�/GLT1-cKO mice.

Effects of short-term treatment with the nuclear exportinhibitor KPT-350 in GLAST �/�/GLT1-cKO miceNucleocytoplasmic transport has a crucial role in cell viability,and the loss of nucleoporins has been reported to impair nuclearimport and export (Roth et al., 2003; Pemberton and Paschal,2005; Bano et al., 2010; Nishimura et al., 2010; Makise et al., 2012;Yamashita et al., 2017a). Because GLAST�/�/GLT1-cKO miceshowed the reduction of Nup153 in motor neurons, we examinedthe changes in the subcellular localization of proteins involved innucleocytoplasmic trafficking. The spinal motor neurons inGLAST�/�/GLT1-cKO mice exhibited normal nuclear immuno-reactivity for KPNB1, which is the member of karyopherinprotein family responsible for protein import, and normal cyto-plasmic immunoreactivity for RanBP1, which regulates the as-sembly and disassembly of karyopherin-cargo complex (Fig.6A,B). In contrast, subcellular localization of CAS protein, knownas exportin-2, was different between controls and GLAST�/�/GLT1-cKO mice. Immunoreactivity for CAS was predominant inthe nucleus of motor neurons in control mice, whereas somespinal motor neurons in GLAST�/�/GLT1-cKO mice showedreduced levels of CAS in the nucleus at 7 weeks of age (earlysymptomatic stage, Fig. 6C). Although the number of CAS-negative motor neurons was low, the percentage of CAS-negativemotor neurons was increased in GLAST�/�/GLT1-cKO mice at 7weeks of age [CAS (�)/ChAT (�) cells (%): control, 2.3 � 1.3%;GLAST�/�/GLT1-cKO, 12.7 � 2.6%; unpaired two-tailed t test,t(5) � 3.21, p � 0.024]. These results indicate that nucleocyto-plasmic transport was slightly impaired in some motor neuronsin GLAST�/�/GLT1-cKO mice.

To examine whether nucleocytoplasmic transport deficits areinvolved in motor dysfunction and motor neuron loss inGLAST�/�/GLT1-cKO mice, we analyzed the effect of KPT-350,a nuclear export inhibitor (Haines et al., 2015), because the invivo toxicity of all the other modifiers of nucleocytoplasmictransport precluded their use. GLAST�/�/GLT1-cKO mice weretreated every other day with KPT-350 (15 mg/kg, orally) startingat 6 weeks of age. GLAST�/�/GLT1-cKO mice treated with KPT-350 delayed motor deficits in the hanging wire test, whereasGLAST�/�/GLT1-cKO mice treated with vehicle showed com-plete hindlimb paralysis [Fig. 6D,E (Fig. 6D: two-way repeated-measures ANOVA for drug � time interaction, F(1,17) � 7.98, p �0.012; post hoc unpaired two-tailed t test, P7W, t(17) � 2.82, p �0.012)]. KPT-350 treatment also delayed motor neuron loss inGLAST�/�/GLT1-cKO mice [Fig. 6F,G (Fig. 6G: unpaired two-tailed t test, t(10) � 4.83, p � 0.00069)]. However, the effects ofKPT-350 were transient, and long-term treatment of KPT-350did not prevent motor neuron loss and motor deficits in GLAST�/�/GLT1-cKO mice (Fig. 7 I,K,L). Although KPT-350 delayed motorneuron death and motor deficits in GLAST�/�/GLT1-cKO mice,the degradation of Nup153 was not prevented [Fig. 6H, I (Fig. 6I:unpaired two-tailed t test, t(4) � 0.13, p � 0.90)].

Together, these findings suggest that nucleocytoplasmic trans-port deficits may partially contribute to excitotoxic spinal motorneuron degeneration in GLAST�/�/GLT1-cKO mice, but may benot a major cause of motor neuron loss in GLAST�/�/GLT1-cKO mice.

Effects of long-term treatment with perampanel, SNJ-1945and KPT-350 in GLAST �/�/GLT1-cKO miceWe next evaluated the efficacy of long-term treatment withperampanel, SNJ-1945, and KPT-350 on the phenotypes ofGLAST�/�/GLT1-cKO mice. GLAST�/�/GLT1-cKO mice weretreated daily with perampanel (20 mg/kg, orally) or SNJ-1945


(200 mg/kg, orally) for 42 d starting at 6 weeks of age. Since thetreatment of control mice with a 15 mg/kg dose of KPT-350 threetimes per week for 3 weeks causes serious weight loss, we orallyadministered KPT-350 (7.5 mg/kg) three times per week from 6to 12 weeks of age. Drug-treated and vehicle-treated animals weremonitored daily for survival and weekly for grip strength. Theresults showed that motor function decline was delayed by theadministration of perampanel or SNJ-1945 [Fig. 7A,E (Fig. 7A:two-way repeated-measures ANOVA for drug � time interac-tion, F(6,204) � 5.59, p � 2.2E-05; post hoc unpaired two-tailed ttests, P7W, t(34) � 2.44, p � 0.020; P8W, t(34) � 3.18, p � 0.0031;P9W, t(34) � 2.70, p � 0.011; P10W, t(34) � 4.28, p � 0.00014;P11W, t(34) � 4.64, p � 5.0E-05; P12W, t(34) � 5.37, p � 5.8E-06;Fig. 7E: two-way repeated-measures ANOVA for drug � timeinteraction, F(6,192) � 7.16, p � 6.6E-07; post hoc unpaired two-tailed t tests, P7W, t(32) � 3.79, p � 0.00062; P8W, t(32) � 2.63,p � 0.013; P9W, t(32) � 2.86, p � 0.0074; P10W, t(32) � 3.64, p �0.00095; P11W, t(32) � 3.88, p � 0.000495; P12W, t(32) � 4.12,p � 0.00025)]. In contrast, KPT-350 treatment delayed motordeficits at 7 weeks of age but not later in GLAST�/�/GLT1-cKOmice [Fig. 7I (two-way repeated-measures ANOVA for drug �time interaction, F(6,186) � 8.85, p � 1.7E-08; post hoc unpairedtwo-tailed t test, P7W, t(31) � 3.03, p � 0.0049)]. SNJ-1945 treat-ment prolonged survival in GLAST�/�/GLT1-cKO mice [Fig. 7F(log rank test, p � 0.026)], whereas KPT-350 treatment had noeffect on survival [Fig. 7J (log rank test, p � 0.86)]. Althoughthere is a tendency for survival rate to be higher in perampanel-treated GLAST �/�/GLT1-cKO mice, there is no significantdifference between perampanel-treated and vehicle-treatedGLAST�/�/GLT1-cKO mice (Fig. 7B (log rank test, p � 0.076)].Consistent with these results, 6 weeks of treatment with peram-panel or SNJ-1945 prevented motor neuron loss in GLAST�/�/GLT1-cKO mice at 12 weeks of age [Fig. 7C,D,G,H (unpairedtwo-tailed t test: Fig. 7D: t(8) � 2.93, p � 0.019; Fig. 7H: t(8) �3.21, p � 0.013)]. On the other hand, the neuroprotective effectof KPT-350 was observed at 7 weeks of age but not at 12 weeks ofage [Figs. 6F,G, 7K,L (Fig. 7L: unpaired two-tailed t test, t(8) �0.01, p � 0.98)]. These results indicate that both perampanel andSNJ-1945 have continued robust protective effects, while the pro-tective effects of KPT-350 in GLAST�/�/GLT1-cKO mice aretransient. Thus, preventing NPC degradation by peramapanel orSNJ-1945 has robust protective effects on motor neurons.

DiscussionIn this study, we generated a new animal model of excitotoxicity:a spinal cord-specific astroglial glutamate transporter (GLT1 andGLAST) double-knock-out mouse. We further demonstratedthat GLAST�/�/GLT1-cKO mice showed selective motor neu-ron loss and severe hindlimb paralysis, which recapitulated thesymptom of motor neuron diseases such as ALS. These findingssuggest that combined loss of astroglial glutamate transportersGLT1 and GLAST is required to induce motor neuron deathleading to paralysis in vivo.

Our results suggest a possible mechanism for spinal motorneuron death that is induced by excitotoxicity (Fig. 8), whichincludes the following: (1) dysfunction or reduction of astroglialglutamate transporters, which increases extracellular glutamate;(2) an increase in glutamate-induced AMPA receptor activation,not NMDA receptor activation, thereby leading to an increase inintracellular Ca 2� levels; (3) calpain hyperactivation by Ca 2�

overload; (4) the degradation of NPCs by calpain; (5) the loss ofnuclear integrity or other unknown mechanisms; and (6) theinduction of motor neuron death (Fig. 8).

GLAST�/�/GLT1-cKO mice exhibited nuclear membraneabnormalities and calpain-mediated degradation of NPCs. Anincrease in the percentage of motor neurons exhibiting nuclearpathology preceded motor neuron death in GLAST�/�/GLT1-cKOmice, suggesting that nuclear pathology is an early pathologicalevent following calpain activation in GLAST�/�/GLT1-cKOmice. In addition, long-term treatment of perampanel and SNJ-1945 has continued beneficial effects in motor neuron loss andthe degradation of Nup153. Consistent with this, a recent studyhas demonstrated that activated calpain degraded NPCs via up-regulation of Ca 2�-permeable AMPA receptors in motor neu-rons of ADAR2 conditional knock-out (AR2) mice (Yamashita etal., 2017a). Together, these results indicate that nuclear mem-brane abnormalities and degradation of NPCs may be involved inmotor neuron death that is driven by AMPA receptor-mediatedexcitotoxicity.

How do nuclear membrane abnormalities and degradation ofNPCs actually lead to motor neuron death? The nucleocytoplas-mic transport through NPCs plays a crucial role in maintainingcell viability and cellular functions (Pemberton and Paschal,2005). Furthermore, previous studies have shown that nucleocy-toplasmic transport deficits due to calpain-dependent degrada-tion of the NPCs were observed in glutamate-treated cultured

Figure 8. Model of AMPA receptor-induced spinal motor neuron death. In spinal cord-specific GLT1 and GLAST double-knock-out mice, the reduction of astroglial glutamate trans-porters (GLAST and GLT1) induces spinal motor neuron death as follows: (1) an increase inextracellular glutamate concentration; (2) an increase in glutamate-induced AMPA receptoractivation, thereby leading to an increase in intracellular Ca 2� levels; (3) the Ca 2� overload-induced activation of calpain; (4) hyperactivated calpain degradation of NPCs; (5) impairmentof nuclear pore function may progress to nuclear “leakiness”; and (6) induces motor neurondeath. Glutamate-induced spinal motor neuron death can be reversed following long-termtreatment with the antiepileptic AMPA receptor antagonist perampanel and the calpain inhib-itor SNJ-1945. AMPAR, AMPA receptor; NMDAR, NMDA receptor; Nup62, Nucleoporin 62;Nup88, Nucleoporin 88; Nup153, Nucleoporin 153.


neurons and AR2 mice (Bano et al., 2010; Yamashita et al.,2017a). In this study, we showed that a selective inhibitor ofnuclear export KPT-350 delayed but did not prevent motor neu-ron death in GLAST�/�/GLT1-cKO mice. In addition, only asmall number of motor neurons showed the change in the sub-cellular localization of CAS in GLAST�/�/GLT1-cKO mice. To-gether, the nucleocytoplasmic transport deficits may transientlycontribute to motor neuron death, but may not be a major down-stream pathway to cause motor neuron death following NPCsdegradation in GLAST�/�/GLT1-cKO mice. Thus, further stud-ies are required to reveal the contribution of nucleocytoplasmictransport defects to motor neuron death and the downstreammechanisms of the NPCs degradation in GLAST�/�/GLT1-cKOmice.

Although our study suggests that the combined loss of glialglutamate transporters is required to recapitulate the ALS-likesymptoms such as motor neuron death leading to paralysis, therewas no report showing a reduction of GLAST expression in ALSpatients. Thus, additional studies are required to evaluate theclinical relevance of GLAST�/�/GLT1-cKO mice to human ALS.

In this study, we demonstrated that motor neuron death waspartially recovered by the pharmacological blockade of AMPAreceptor and calpain activation, which could inhibit the degrada-tion of Nup153. Therefore, our findings suggest that perampaneland SNJ-1945 could be a novel therapeutic strategy for manyacute and chronic neurological diseases that involve AMPAreceptor-mediated excitotoxicity, such as ischemic or traumaticinjury and ALS.

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